Products of Selenite/Thiols Interaction Have Reducing Properties, Cleave Plasmid DNA and Decrease Rat Blood Pressure and Tension of Rat Mesenteric Artery.

Selenium compounds exert their antioxidant activity mostly when the selenium atom is incorporated into selenoproteins. In our work, we tested the possibility that selenite itself interacts with thiols to form active species that have reducing properties. Therefore, we studied the reduction of 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide radical (•cPTIO), damage of plasmid DNA (pDNA), modulation of rat hemodynamic parameters and tension of isolated arteries induced by products of interaction of selenite with thiols. We found that the products of selenite interaction with thiols had significant reducing properties that could be attributed mainly to the selenide and that selenite had catalytic properties in the access of thiols. The potency of thiols to reduce •cPTIO in the interaction with selenite was cysteine > homocysteine > glutathione reduced > N-acetylcysteine. Thiol/selenite products cleaved pDNA, with superoxide dismutase enhancing these effects suggesting a positive involvement of superoxide anion in the process. The observed •cPTIO reduction and pDNA cleavage were significantly lower when selenomethionine was used instead of selenite. The products of glutathione/selenite interaction affected several hemodynamic parameters including rat blood pressure decrease. Notably, the products relaxed isolated mesenteric artery, which may explain the observed decrease in rat blood pressure. In conclusion, we found that the thiol/selenite interaction products exhibited significant reducing properties which can be used in further studies of the treatment of pathological conditions caused by oxidative stress. The results of decreased rat blood pressure and the tension of mesenteric artery may be perspective in studies focused on cardiovascular disease and their prevention.

Use of a rat model to characterize 35 arterial pulse wave parameters in a comparative study of isoflurane and Zoletil/xylazine anesthesia and the effect of Acanthopanax senticosus extract.

BACKGROUND: Information obtained from arterial pulse waveforms (APW) can be useful for characterizing the cardiovascular system. To achieve this, it is necessary to know the detailed characteristics of APWs in different states of an organism, which would allow APW parameters (APW-Ps) to be assigned to particular (patho)physiological conditions. Therefore, our work aimed to characterize 35 APW-Ps in rats under the influence of isoflurane (ISO) and Zoletil/xylazine (ZO/XY) anesthesia and to study the effect of root extract from Acanthopanax senticosus (ASRE) in these anesthetic conditions. METHODS: The right jugular vein of anesthetized rats was cannulated for the administration of ASRE and the left carotid artery for the detection of APWs from which 35 APW-Ps were evaluated. RESULTS: We obtained data on 35 APW-Ps, which significantly depended on the anesthesia, and thus, they characterized the cardiovascular system under these two conditions. ASRE transiently modulated all 35 APW-Ps, including a transient decrease in systolic and diastolic blood pressure (BP) and heart rate or increases in pulse BP, dP/dtmax , and systolic and diastolic areas. Whereas the transient effects of ASRE were similar, the extract had prolonged disturbing effects on the cardiovascular system in rats under ZO/XY but not under ISO anesthesia. This negative effect can result from the disturbance caused by ZO/XY anesthesia on the cardiovascular system. CONCLUSIONS: We characterized 35 APW-Ps of rats under ISO and ZO/XY anesthesia and found that ASRE contains compounds that can modulate the properties of the cardiovascular system, which significantly depended on the status of the cardiovascular system. This should be considered when using ASRE as a nutritional supplement by individuals with cardiovascular problems.

The Phthalic Selenoanhydride Decreases Rat Blood Pressure and Tension of Isolated Mesenteric, Femoral and Renal Arteries.

Phthalic selenoanhydride (R-Se) solved in physiological buffer releases various reactive selenium species including H2Se. It is a potential compound for Se supplementation which exerts several biological effects, but its effect on the cardiovascular system is still unknown. Therefore, herein we aimed to study how R-Se affects rat hemodynamic parameters and vasoactive properties in isolated arteries. The right jugular vein of anesthetized Wistar male rats was cannulated for IV administration of R-Se. The arterial pulse waveform (APW) was detected by cannulation of the left carotid artery, enabling the evaluation of 35 parameters. R-Se (1-2 µmol kg-1), but not phthalic anhydride or phthalic thioanhydride, transiently modulated most of the APW parameters including a decrease in systolic and diastolic blood pressure, heart rate, dP/dtmax relative level, or anacrotic/dicrotic notches, whereas systolic area, dP/dtmin delay, dP/dtd delay, anacrotic notch relative level or its delay increased. R-Se (~10-100 µmol L-1) significantly decreased the tension of precontracted mesenteric, femoral, and renal arteries, whereas it showed a moderate vasorelaxation effect on thoracic aorta isolated from normotensive Wistar rats. The results imply that R-Se acts on vascular smooth muscle cells, which might underlie the effects of R-Se on the rat hemodynamic parameters.

Extract of Acanthopanax senticosus and Its Components Interacting with Sulfide, Cysteine and Glutathione Increase Their Antioxidant Potencies and Inhibit Polysulfide-Induced Cleavage of Plasmid DNA.

Aqueous root extract from Acanthopanax senticosus (ASRE) has a wide range of medicinal effects. The present work was aimed at studying the influence of sulfide, cysteine and glutathione on the antioxidant properties of ASRE and some of its selected phytochemical components. Reduction of the 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide (●cPTIO) stable radical and plasmid DNA (pDNA) cleavage in vitro assays were used to evaluate antioxidant and DNA-damaging properties of ASRE and its individual components. We found that the interaction of ASRE and its two components, caffeic acid and chlorogenic acid (but not protocatechuic acid and eleutheroside B or E), with H2S/HS-, cysteine or glutathione significantly increased the reduction of the ●cPTIO radical. In contrast, the potency of ASRE and its selected components was not affected by Na2S4, oxidized glutathione, cystine or methionine, indicating that the thiol group is a prerequisite for the promotion of the antioxidant effects. ASRE interacting with H2S/HS- or cysteine displayed a bell-shaped effect in the pDNA cleavage assay. However, ASRE and its components inhibited pDNA cleavage induced by polysulfides. In conclusion, we suggest that cysteine, glutathione and H2S/HS- increase antioxidant properties of ASRE and that changes of their concentrations and the thiol/disulfide ratio can influence the resulting biological effects of ASRE.

EPR Study of KO2 as a Source of Superoxide and •BMPO-OH/OOH Radical That Cleaves Plasmid DNA and Detects Radical Interaction with H2S and Se-Derivatives.

Superoxide radical anion (O2•-) and its derivatives regulate numerous physiological and pathological processes, which are extensively studied. The aim of our work was to utilize KO2 as a source of O2•- and the electron paramagnetic resonance (EPR) spin trapping 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) technique for the preparation of •BMPO-OOH and/or •BMPO-OH radicals in water solution without DMSO. The method distinguishes the interactions of various compounds with •BMPO-OOH and/or •BMPO-OH radicals over time. Here, we show that the addition of a buffered BMPO-HCl mixture to powdered KO2 formed relatively stable •BMPO-OOH and •BMPO-OH radicals and H2O2, where the •BMPO-OOH/OH ratio depended on the pH. At a final pH of ~6.5-8.0, the concentration of •BMPO-OOH radicals was ≥20 times higher than that of •BMPO-OH, whereas at pH 9.0-10.0, the •BMPO-OH radicals prevailed. The •BMPO-OOH/OH radicals effectively cleaved the plasmid DNA. H2S decreased the concentration of •BMPO-OOH/OH radicals, whereas the selenium derivatives 1-methyl-4-(3-(phenylselanyl) propyl) piperazine and 1-methyl-4-(4-(phenylselanyl) butyl) piperazine increased the proportion of •BMPO-OH over the •BMPO-OOH radicals. In conclusion, the presented approach of using KO2 as a source of O2•-/H2O2 and EPR spin trap BMPO for the preparation of •BMPO-OOH/OH radicals in a physiological solution could be useful to study the biological effects of radicals and their interactions with compounds.

The impact of gut microbiota metabolites on cellular bioenergetics and cardiometabolic health.

Recent research demonstrates a reciprocal relationship between gut microbiota-derived metabolites and the host in controlling the energy homeostasis in mammals. On the one hand, to thrive, gut bacteria exploit nutrients digested by the host. On the other hand, the host utilizes numerous products of gut bacteria metabolism as a substrate for ATP production in the colon. Finally, bacterial metabolites seep from the gut into the bloodstream and interfere with the host's cellular bioenergetics machinery. Notably, there is an association between alterations in microbiota composition and the development of metabolic diseases and their cardiovascular complications. Some metabolites, like short-chain fatty acids and trimethylamine, are considered markers of cardiometabolic health. Others, like hydrogen sulfide and nitrite, demonstrate antihypertensive properties. Scientific databases were searched for pre-clinical and clinical studies to summarize current knowledge on the role of gut microbiota metabolites in the regulation of mammalian bioenergetics and discuss their potential involvement in the development of cardiometabolic disorders. Overall, the available data demonstrates that gut bacteria products affect physiological and pathological processes controlling energy and vascular homeostasis. Thus, the modulation of microbiota-derived metabolites may represent a new approach for treating obesity, hypertension and type 2 diabetes.

Cardiovascular "Patterns" of H2S and SSNO--Mix Evaluated from 35 Rat Hemodynamic Parameters.

This work is based on the hypothesis that it is possible to characterize the cardiovascular system just from the detailed shape of the arterial pulse waveform (APW). Since H2S, NO donor S-nitrosoglutathione (GSNO) and their H2S/GSNO products (SSNO--mix) have numerous biological actions, we aimed to compare their effects on APW and to find characteristic "patterns" of their actions. The right jugular vein of anesthetized rats was cannulated for i.v. administration of the compounds. The left carotid artery was cannulated to detect APW. From APW, 35 hemodynamic parameters (HPs) were evaluated. H2S transiently influenced all 35 HPs and from their cross-relationships to systolic blood pressure "patterns" and direct/indirect signaling pathways of the H2S effect were proposed. The observed "patterns" were mostly different from the published ones for GSNO. Effect of SSNO--mix (≤32 nmol kg-1) on blood pressure in the presence or absence of a nitric oxide synthase inhibitor (L-NAME) was minor in comparison to GSNO, suggesting that the formation of SSNO--mix in blood diminished the hemodynamic effect of NO. The observed time-dependent changes of 35 HPs, their cross-relationships and non-hysteresis/hysteresis profiles may serve as "patterns" for the conditions of a transient decrease/increase of blood pressure caused by H2S.

•BMPO-OOH Spin-Adduct as a Model for Study of Decomposition of Organic Hydroperoxides and the Effects of Sulfide/Selenite Derivatives. An EPR Spin-Trapping Approach.

Lipid hydroperoxides play an important role in various pathophysiological processes. Therefore, a simple model for organic hydroperoxides could be helpful to monitor the biologic effects of endogenous and exogenous compounds. The electron paramagnetic resonance (EPR) spin-trapping technique is a useful method to study superoxide (O2•-) and hydroxyl radicals. The aim of our work was to use EPR with the spin trap 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO), which, by trapping O2•- produces relatively stable •BMPO-OOH spin-adduct, a valuable model for organic hydroperoxides. We used this experimental setup to investigate the effects of selected sulfur/selenium compounds on •BMPO-OOH and to evaluate the antioxidant potential of these compounds. Second, using the simulation of time-dependent individual BMPO adducts in the experimental EPR spectra, the ratio of •BMPO-OH/•BMPO-OOH-which is proportional to the transformation/decomposition of •BMPO-OOH-was evaluated. The order of potency of the studied compounds to alter •BMPO-OOH concentration estimated from the time-dependent •BMPO-OH/•BMPO-OOH ratio was as follows: Na2S4 > Na2S4/SeO32- > H2S/SeO32- > Na2S2 ~Na2S2/SeO32- ~H2S > SeO32- ~SeO42- ~control. In conclusion, the presented approach of the EPR measurement of the time-dependent ratio of •BMPO-OH/•BMPO-OOH could be useful to study the impact of compounds to influence the transformation of •BMPO-OOH.

Characterization of Rat Cardiovascular System by Anacrotic/Dicrotic Notches in the Condition of Increase/Decrease of NO Bioavailability.

We characterized modes of action of NO-donor S-nitrosoglutathione (GSNO) and NO-synthase inhibitor l-NAME derived from dicrotic (DiN) and anacrotic (AnN) notches of rat arterial pulse waveform (APW) in the condition of increased/decreased NO bioavailability. The cross-relationship patterns of DiN and AnN with 34 hemodynamic parameters (HPs) induced by GSNO and l-NAME are presented. After GSNO bolus administration, approximate non-hysteresis relationships were observed in the difference between DiN-AnN (mmHg) blood pressure (BP) and other 19 HPs, suggesting that these HPs, i.e., their signaling pathways, responding to NO concentration, are directly connected. Hysteresis relationships were observed between DiN-AnN (mmHg) and other 14 HPs, suggesting that signaling pathways of these HPs are indirectly connected. The hysteresis relationships were only observed between the time interval DiN-AnN (ms) and other 34 HPs, indicating no direct connection of signaling pathways. The cross-relationship patterns of DiN-AnN (mmHg), but not DiN-AnN (ms), induced by l-NAME were in accordance to the increased NO bioavailability induced by GSNO. In conclusion, we found the non-hysteresis/hysteresis cross-relationship "patterns" of DiN-AnN intervals to other HPs in the presence of GSNO that revealed their direct or indirect signaling pathways connections. This may contribute to our understanding of biological effects of natural substances that modulate NO production and/or NO signaling pathways.

Patterns and Direct/Indirect Signaling Pathways in Cardiovascular System in the Condition of Transient Increase of NO.

AIM: To study "patterns" and connections of signaling pathways derived from the rat arterial pulse waveform (APW) under the condition of transient NO increase. METHODS AND RESULTS: The right jugular vein of anesthetized Wistar rats was cannulated for administration of NO donor S-nitrosoglutathione. The left carotid artery was cannulated to detect APW. From rat APW, 35 hemodynamic parameters (HPs) and several their crossrelationships were evaluated. We introduced a new methodology to study "patterns" and connections of different signaling pathways, which are suggested from hysteresis and nonhysteresis crossrelationships of 35 rat HPs. Here, we show parallel time-dependent patterns of 35 HPs and some of their crossrelationships under the condition of transient increase of NO bioavailability by administration of S-nitrosoglutathione. Approximate nonhysteresis relationships were observed between systolic blood pressure and at least 11 HPs suggesting that these HPs, i.e., their signaling pathways, responding to NO concentration, are directly connected. Hysteresis relationships were observed between systolic blood pressure and at least 14 HPs suggesting that the signaling pathways of these HPs are indirectly connected. Totally, from the crossrelationships of 35 HPs, one can obtain 595 "patterns" and indication of direct or indirect connections between the signaling pathways. CONCLUSION: We described the procedure leading virtually to 595 relationships, from which "patterns" for transient NO increase and direct or indirect connections of signaling pathways can be suggested. From a clinical perspective, this approach may be used in animal models and in humans to create a data bank of patterns of crossrelationships of HPs for different cardiovascular conditions. By comparison with unknown patterns of studied APW with the data bank patterns, it would be possible to determine cardiovascular conditions of the studied subject from the recorded arterial blood pressure. Additionally, it can help to find molecular mechanism of particular (patho-) physiological conditions or drug action and may have predictive or diagnostic value.

Mathematical relationships of patterns of 35 rat haemodynamic parameters for conditions of hypertension resulting from decreased nitric oxide bioavailability.

NEW FINDINGS: What is the central question of this study? Can the cross-relationship between 35 rat arterial pulse waveform (APW) parameters be described by known mathematical functions and can mathematical parameters be obtained for conditions in a model of hypertension resulting from decreased NO bioavailability? What is the main finding and its importance? Mathematical functions and their parameters were obtained that approximate the cross-relationships of 35 APW parameters to systolic blood pressure and to the augmentation index in conditions of decreased NO bioavailability. The results enable APW parameters to be assigned to decreased NO bioavailability, which may have predictive or diagnostic value. ABSTRACT: Information obtained from the arterial pulse waveform (APW) using haemodynamic parameters (HPs) is useful for characterization of the cardiovascular system in particular (patho)physiological conditions. Our goal was to find out whether the relationships between rat HPs could be described by simple mathematical functions and to find mathematical parameters for conditions of high blood pressure (BP) resulting from decreased NO bioavailability. The right jugular vein of anaesthetized Wistar rats was cannulated for i.v. administration of Nω -nitro-l-arginine methyl ester (l-NAME). The left common carotid artery was cannulated to detect the APW. From 10 points on the rat APW we defined 35 HPs (some were known already) and found 595 cross-relationships between HPs showing unique patterns for particular cardiovascular conditions. Here we show parallel time-dependent changes of 35 HPs and some of their cross-relationships in condition of high BP induced by l-NAME. We found that most of the time-dependent changes of 35 HPs and their relationships were very well fitted by simple mathematical functions, e.g. a linear function, exponential growth, exponential decay or exponential rise to maximum. The results may enable the mathematical functions to be assigned for decreased NO bioavailability, which may have predictive or diagnostic value for conditions of high BP. Using this approach, it may be possible to find unique cross-relationship patterns of HPs and mathematical functions between HPs for different cardiovascular (patho)physiological or drug-modulating conditions. This knowledge can be used in studying the molecular mechanisms of particular (patho)physiological conditions or drug actions and may have predictive or diagnostic value.

Products of Sulfide/Selenite Interaction Possess Antioxidant Properties, Scavenge Superoxide-Derived Radicals, React with DNA, and Modulate Blood Pressure and Tension of Isolated Thoracic Aorta.

Selenium (Se), an essential trace element, and hydrogen sulfide (H2S), an endogenously produced signalling molecule, affect many physiological and pathological processes. However, the biological effects of their mutual interaction have not yet been investigated. Herein, we have studied the biological and antioxidant effects of the products of the H2S (Na2S)/selenite (Na2SeO3) interaction. As detected by the UV-VIS and EPR spectroscopy, the product(s) of the H2S-Na2SeO3 and H2S-SeCl4 interaction scavenged superoxide-derived radicals and reduced ·cPTIO radical depending on the molar ratio and the preincubation time of the applied interaction mixture. The results confirmed that the transient species are formed rapidly during the interaction and exhibit a noteworthy biological activity. In contrast to H2S or selenite acting on their own, the H2S/selenite mixture cleaved DNA in a bell-shaped manner. Interestingly, selenite protected DNA from the cleavage induced by the products of H2S/H2O2 interaction. The relaxation effect of H2S on isolated thoracic aorta was eliminated when the H2S/selenite mixture was applied. The mixture inhibited the H2S biphasic effect on rat systolic and pulse blood pressure. The results point to the antioxidant properties of products of the H2S/selenite interaction and their effect to react with DNA and influence cardiovascular homeostasis. The effects of the products may contribute to explain some of the biological effects of H2S and/or selenite, and they may imply that a suitable H2S/selenite supplement might have a beneficial effect in pathological conditions arisen, e.g., from oxidative stress.

Type 1 Sodium Calcium Exchanger Forms a Complex with Carbonic Anhydrase IX and Via Reverse Mode Activity Contributes to pH Control in Hypoxic Tumors.

Hypoxia and acidosis are among the key microenvironmental factors that contribute to cancer progression. We have explored a possibility that the type 1Na+/Ca2+ exchanger (NCX1) is involved in pH control in hypoxic tumors. We focused on changes in intracellular pH, co-localization of NCX1, carbonic anhydrase IX (CA IX), and sodium proton exchanger type 1 (NHE1) by proximity ligation assay, immunoprecipitation, spheroid formation assay and migration of cells due to treatment with KB-R7943, a selective inhibitor of the reverse-mode NCX1. In cancer cells exposed to hypoxia, reverse-mode NCX1 forms a membrane complex primarily with CA IX and also with NHE1. NCX1/CA IX/NHE1 assembly operates as a metabolon with a potent ability to extrude protons to the extracellular space and thereby facilitate acidosis. KB-R7943 prevents formation of this metabolon and reduces cell migration. Thus, we have shown that in hypoxic cancer cells, NCX1 operates in a reverse mode and participates in pH regulation in hypoxic tumors via cooperation with CAIX and NHE1.

In Vivo Measurement of H2S, Polysulfides, and "SSNO- Mix"-Mediated Vasoactive Responses and Evaluation of Ten Hemodynamic Parameters from Rat Arterial Pulse Waveform.

The chapter describes protocols and pitfalls in in vivo studies of drug effects on anesthetized rats. It focuses on the preparation of Na2S, Na2S4, and "SSNO- mix" solutions for rat intravenous administration, surgical preparation of jugular vein for drug administration, and preparation of carotid and tail arteries for recording of arterial pulse waveform (APW) at high resolution. It describes evaluation of ten hemodynamic parameters from APW and measurement of apparent pulse wave velocity.

Inorganic Polysulfides and Related Reactive Sulfur­Selenium Species from the Perspective of Chemistry.

Polysulfides (H2Sx) represent a class of reactive sulfur species (RSS) which includes molecules such as H2S2, H2S3, H2S4, and H2S5, and whose presence and impact in biological systems, when compared to other sulfur compounds, has only recently attracted the wider attention of researchers. Studies in this field have revealed a facet-rich chemistry and biological activity associated with such chemically simple, still unusual inorganic molecules. Despite their chemical simplicity, these inorganic species, as reductants and oxidants, metal binders, surfactant-like "cork screws" for membranes, components of perthiol signalling and reservoirs for inorganic hydrogen sulfide (H2S), are at the centre of complicated formation and transformation pathways which affect numerous cellular processes. Starting from their chemistry, the hidden presence and various roles of polysulfides in biology may become more apparent, despite their lack of clear analytical fingerprints and often murky biochemical footprints. Indeed, the biological chemistry of H2Sx follows many unexplored paths and today, the relationship between H2S and its oxidized H2Sx species needs to be clarified as a matter of "unmistaken identity". Simultaneously, emerging species, such as HSSeSH and SenS8-n, also need to be considered in earnest.

Sulfide (Na2S) and Polysulfide (Na2S2) Interacting with Doxycycline Produce/Scavenge Superoxide and Hydroxyl Radicals and Induce/Inhibit DNA Cleavage.

Doxycycline (DOXY) is an antibiotic routinely prescribed in human and veterinary medicine for antibacterial treatment, but it has also numerous side effects that include oxidative stress, inflammation, cancer or hypoxia-induced injury. Endogenously produced hydrogen sulfide (H2S) and polysulfides affect similar biological processes, in which reactive oxygen species (ROS) play a role. Herein, we have studied the interaction of DOXY with H2S (Na2S) or polysulfides (Na2S2, Na2S3 and Na2S4) to gain insights into the biological effects of intermediates/products that they generate. To achieve this, UV-VIS, EPR spectroscopy and plasmid DNA (pDNA) cleavage assay were employed. Na2S or Na2S2 in a mixture with DOXY, depending on ratio, concentration and time, displayed bell-shape kinetics in terms of producing/scavenging superoxide and hydroxyl radicals and decomposing hydrogen peroxide. In contrast, the effects of individual compounds (except for Na2S2) were hardly observable. In addition, DOXY, as well as oxytetracycline and tetracycline, interacting with Na2S or other studied polysulfides reduced the •cPTIO radical. Tetracyclines induced pDNA cleavage in the presence of Na2S. Interestingly, they inhibited pDNA cleavage induced by other polysulfides. In conclusion, sulfide and polysulfides interacting with tetracyclines produce/scavenge free radicals, indicating a consequence for free radical biology under conditions of ROS production and tetracyclines administration.

Sodium/calcium exchanger is involved in apoptosis induced by H2S in tumor cells through decreased levels of intracellular pH.

We explored possibility that sodium/calcium exchanger 1 (NCX1) is involved in pH modulation and apoptosis induction in GYY4137 treated cells. We have shown that although 10 days treatment with GYY4137 did not significantly decreased volume of tumors induced by colorectal cancer DLD1 cells in nude mice, it already induced apoptosis in these tumors. Treatment of DLD1 and ovarian cancer A2780 cells with GYY4137 resulted in intracellular acidification in a concentration-dependent manner. We observed increased mRNA and protein expression of both, NCX1 and sodium/hydrogen exchanger 1 (NHE1) in DLD1-induced tumors from GYY4137-treated mice. NCX1 was coupled with NHE1 in A2780 and DLD1 cells and this complex partially disintegrated after GYY4137 treatment. We proposed that intracellular acidification is due to uncoupling of NCX1/NHE1 complex rather than blocking of the reverse mode of NCX1, probably due to internalization of NHE1. Results might contribute to understanding molecular mechanism of H2S-induced apoptosis in tumor cells.

Endogenous H2S producing enzymes are involved in apoptosis induction in clear cell renal cell carcinoma.

BACKGROUND: Knowledge about the expression and thus a role of enzymes that produce endogenous H2S - cystathionine-ß-synthase, cystathionine γ-lyase and mercaptopyruvate sulfurtransferase - in renal tumors is still controversial. In this study we aimed to determine the expression of these enzymes relatively to the expression in unaffected part of kidney from the same patient and to found relation of these changes to apoptosis. To evaluate patient's samples, microarray and immunohistochemistry was used. METHODS: To determine the physiological importance, we used RCC4 stable cell line derived from clear cell renal cell carcinoma, where apoptosis induction by a mixture of five chemotherapeutics with/without silencing of H2S-producing enzymes was detected. Immunofluorescence was used to determine each enzyme in the cells. RESULTS: In clear cell renal cell carcinomas, expression of H2S-producing enzymes was mostly decreased compared to a part of kidney that was distal from the tumor. To evaluate a potential role of H2S-producing enzymes in the apoptosis induction, we used RCC4 stable cell line. We have found that silencing of cystathionine-ß-synthase and cystathionine γ-lyase prevented induction of apoptosis. Immunofluorescence staining clearly showed that these enzymes were upregulated during apoptosis in RCC4 cells. CONCLUSION: Based on these results we concluded that in clear cell renal cell carcinoma, reduced expression of the H2S-producing enzymes, mainly cystathionine γ-lyase, might contribute to a resistance to the induction of apoptosis. Increased production of the endogenous H2S, or donation from the external sources might be of a therapeutic importance in these tumors.

Polysulfides and products of H2S/S-nitrosoglutathione in comparison to H2S, glutathione and antioxidant Trolox are potent scavengers of superoxide anion radical and produce hydroxyl radical by decomposition of H2O2.

Exogenous and endogenously produced sulfide derivatives, such as H2S/HS-/S2-, polysulfides and products of the H2S/S-nitrosoglutathione interaction (S/GSNO), affect numerous biological processes in which superoxide anion (O2-) and hydroxyl (OH) radicals play an important role. Their cytoprotective-antioxidant and contrasting pro-oxidant-toxic effects have been reported. Therefore, the aim of our work was to contribute to resolving this apparent inconsistency by studying sulfide derivatives/free radical interactions and their consequent biological effects compared to the antioxidants glutathione (GSH) and Trolox. Using the electron paramagnetic resonance (EPR) spin trapping technique and O2-, we found that a polysulfide (Na2S4) and S/GSNO were potent scavengers of O2- and cPTIO radicals compared to H2S (Na2S), GSH and Trolox, and S/GSNO scavenged the DEPMPO-OH radical. As detected by the EPR spectra of DEPMPO-OH, the formation of OH in physiological solution by S/GSNO was suggested. All the studied sulfide derivatives, but not Trolox or GSH, had a bell-shaped potency to decompose H2O2 and produced OH in the following order: S/GSNO > Na2S4 ≥ Na2S > GSH = Trolox = 0, but they scavenged OH at higher concentrations. In studies of the biological consequences of these sulfide derivatives/H2O2 properties, we found the following: (i) S/GSNO alone and all sulfide derivatives in the presence of H2O2 cleaved plasmid DNA; (ii) S/GSNO interfered with viral replication and consequently decreased the infectivity of viruses; (iii) the sulfide derivatives induced apoptosis in A2780 cells but inhibited apoptosis induced by H2O2; and (iv) Na2S4 modulated intracellular calcium in A87MG cells, which depended on the order of Na2S4/H2O2 application. We suggest that the apparent inconsistency of the cytoprotective-antioxidant and contrasting pro-oxidant-toxic biological effects of sulfide derivatives results from their time- and concentration-dependent radical production/scavenging properties and their interactions with O2-, OH and H2O2. The results imply a direct involvement of sulfide derivatives in O2- and H2O2/OH free radical pathways modulating antioxidant/toxic biological processes.

Parenteral Na2S, a fast-releasing H2S donor, but not GYY4137, a slow-releasing H2S donor, lowers blood pressure in rats.

Hydrogen sulfide (H2S) is involved in blood pressure regulation. We evaluated hemodynamic effects of Na2S and morpholin-4-ium (4-methoxyphenyl)(morpholino)phosphinodithioate (GYY4137), H2S donors. GYY4137 is the most widely studied slow-releasing H2S donor, however, its ability to release H2S under physiological conditions is unclear. Hemodynamics were recorded in anaesthetized Wistar-Kyoto rats at baseline and after intravenous (IV) or intraperitoneal (IP) administration of either a vehicle (20% dimethyl sulfoxide), GYY4137 or Na2S. The stability of GYY4137 in buffers and in plasma was evaluated with nuclear magnetic resonance. The vehicle, as well as GYY4137, given IV did not affect mean arterial blood pressure (MABP), whereas Na2S produced a significant decrease in MABP. Similarly, IP given Na2S, but not GYY4137, lowered MABP. In the buffers at pH of 7.4 and 5.5 and in rat plasma no reaction of GYY4137 was found during 18 hours of observation. In contrast, rapid decomposition of GYY4137 occurred in buffers at pH 2.0. In conclusion, parenteral GYY4137 does not exert a hemodynamic effect in Wistar-Kyoto rats. This seems to be due to the high stability of GYY4137 at physiological pH. Therefore, it is likely that widely reported biological effects of GYY4137 are not H2S-dependent but may depend on GYY4137 itself. However, the H2S-dependent biological effects of GYY4137 may be expected in tissues characterized by low pH.